1. Field of the Invention
The present invention relates to an inspection method permitting efficient observation or inspection of a liquid sample containing cultured cells or the like. The invention also relates to a reagent solution used in this inspection method.
2. Description of Related Art
Living organisms including human beings are multicellular animals. Living organisms develop diseases if information cannot be transmitted normally among cells or if viruses or chemical substances cling to cells. For this reason, in the fields of molecular biology and pharmaceutics, research is conducted by peeling off cells from a living organism, cultivating the cells on a laboratory dish, giving a stimulus, such as electricity, chemical substance, or medicine to the cells, and observing the resulting reaction on the cellular level.
In the past, optical microscopes have been used for such observation. Manipulators or pipettes have been employed to give stimuli to cells. Frequently, important portions to be observed are very tiny regions of less than 0.1 μm that are impossible to observe with an optical microscope. For example, diseases arising from the inability to exchange substances normally among biological cells include hypertension, diabetes insipidus, arrhythmia, myopathy, diabetes, and deprementia. Exchange of substances among cells is performed by ion channels having sizes of about 10 nm existing in cell membranes. Because it is difficult to observe such ion channels with optical microscopes, there has been a demand for a technique enabling observation using a scanning electron microscope (SEM) having high resolution.
However, a sample to be inspected with an inspection apparatus incorporating SEM capabilities is normally placed in a sample chamber whose inside pressure has been reduced by vacuum pumping. The sample placed in the sample chamber, which, in turn, is placed in a reduced-pressure ambient in this way, is irradiated with an electron beam (charged-particle beam). Secondary signals, such as secondary electrons or backscattered electrons, produced from the sample in response to the irradiation are detected.
In such inspection of a sample using SEM, the sample is exposed to a reduced-pressure ambient. Therefore, moisture evaporates from the sample, so that the cells die. It has been impossible to observe reactions of living cells to a stimulus.
Accordingly, when an inspection is performed under the condition where the sample contains moisture, it is necessary to prevent the sample from being exposed to the reduced-pressure ambient; otherwise, moisture would evaporate from the sample. One conceivable method of inspecting a sample using SEM without exposing the sample to a reduced-pressure ambient in this way consists of preparing a sample holder (sample capsule that may or may not be hermetically sealed) whose opening (aperture) has been sealed off by a film, placing the sample in the holder, and installing the holder in an SEM sample chamber that is placed in the reduced-pressure ambient.
The inside of the sample holder in which the sample is placed is not evacuated. The film that covers the opening formed in the sample holder (sample capsule) can withstand the pressure difference between the reduced-pressure ambient inside the SEM sample chamber and the ambient (e.g., atmospheric-pressure ambient) of the inside of the sample holder that is not pumped down. Furthermore, the film permits an electron beam to pass therethrough (see JP-T-2004-515049).
When a sample is inspected, a culture medium is first put into a sample capsule together with cells. The cells are cultivated on the film. Then, the sample capsule is placed into an SEM sample chamber that is in a reduced-pressure ambient. An electron beam is directed at the sample placed within the sample capsule from outside the capsule via the film on the capsule placed in the SEM sample chamber. Backscattered electrons are produced from the irradiated sample. The backscattered electrons pass through the film on the capsule and are detected by a backscattered electron detector mounted in the SEM sample chamber. Consequently, an SEM image is derived.
However, with this technique, the sample is sealed in the closed space and so it has been impossible to give a stimulus to cells using a manipulator or pipette. The amount of the culture medium put into the sample capsule is about 15 μl. Therefore, as the culture medium evaporates, the salinity concentration rises, making it difficult to culture cells. Where the cells should be observed or inspected in vivo, there arises a problem.
This problem can be solved by increasing the size of the sample capsule to increase the capacity. However, if the film is destroyed either by a stimulation induced by an electron beam or by a mechanical stimulus, a new problem is created. That is, the inside of the apparatus is contaminated with a large amount of culture medium.
An example of a method of obtaining an SEM image by preparing a film withstanding the pressure difference between vacuum and atmospheric pressure, irradiating a sample with an electron beam via the film, and detecting backscattered electrons produced from the sample in this way is described also in “Atmospheric scanning electron microscopy,” Green, Evan Drake Harriman, Ph.D., Stanford University, 1993 (especially, Chapter 1: Introduction).
Examples in which two films of the structure described above are placed opposite to each other with a sample interposed between the films and in which an image is acquired by a transmission electron microscope are described in JP-A-47-24961 and JP-A-6-318445. Especially, the latter patent document also states a case in which an SEM image of the sample interposed between such films is acquired.
JP-A-2007-292702 discloses a sample inspection apparatus equipped with an open-close valve for partitioning the space between a film and primary beam irradiation device within a vacuum chamber in order to permit the sample held on the film to be exchanged quickly and to prevent contamination inside the vacuum chamber.
The resolution of an optical microscope is not high enough to observe very tiny regions of biological cells. Imaging using SEM is required. In order to observe cells by SEM while maintaining the liquid, a sample (cells) cultured on a laboratory dish is sealed into a sample capsule. The sample is irradiated with an electron beam via the film formed on the sample capsule. Thus, the sample is imaged.
However, the sample capsule is a narrow, closed space. Therefore, there is the problem that it has been impossible to directly observe the state of the sample immediately after a stimulus is given from the outside to the sample using a manipulator or pipette. Furthermore, the capacity inside the sample capsule is small. Consequently, when moisture evaporates and the salinity concentration rises, it is difficult to culture cells for a long time inside the sample capsule. Hence, there are problems in observing cells for a long time.
JP-A-2007-292702 states that when a sample is exchanged, the space between the film and the primary beam irradiation device is partitioned off by the open-close valve and that under this condition, only the space on the film side is returned to the normal pressure. It also states that if the film is damaged during inspection of the sample, the valve is closed, partitioning off the space inside the vacuum chamber to thereby prevent contamination of the inside of the vacuum chamber.
With the open-close valve described in JP-A-2007-292702, however, the space inside the vacuum chamber is partitioned off. Therefore, it is possible to prevent contamination of the space in which the primary beam irradiation device is located, but it has been difficult to prevent contamination of the space on the film side.